Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow.

Studies of the metal content of metalloproteins in tissues from the human central nervous system (CNS) can be compromised by preparative techniques which alter levels of, or interactions between, metals and the protein of interest within a complex mixture. We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and either proton or synchrotron X-ray fluorescence within electrophoresis gels to analyze the endogenous metal content of copper-zinc superoxide dismutase (SOD1) purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis and Parkinson's disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. Validating our novel approach, we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein versus confounding metalloproteins. We analyzed tissues from nine healthy individuals and five CNS regions (occipital cortex, substantia nigra, locus coeruleus, dorsal spinal cord, and ventral spinal cord). We found that Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28, a ratio very close to the expected value of 1. Our methodological workflow can be applied to the study of endogenous native SOD1 in a pathological context and adapted to a range of metalloproteins from human tissues and other sources.

Sub-micrometer Nanosecond LA-ICP-MS Imaging at Pixel Acquisition Rates above 250 Hz via a Low-Dispersion Setup.

The analytical figures of merit of a low-dispersion (ultrafast) ablation cell geometry within the Cobalt ablation chamber, integrated into a nanosecond laser ablation-inductively coupled plasma-mass spectrometer system, are reported. The system was investigated for its capability for fast high-resolution elemental imaging. A spot of 0.6 µm diameter was achieved on the sample surface by aperture imaging of a 10 µm pinhole. The resulting conical crater (0.6 µm ⌀ × 130 nm↓) morphology in a Au-coated glass target and carbon-coated silica wafer was characterized with atomic force microscopy. The Cobalt ablation chamber is based around a motorized height-adjustable tube cell, which allows modulating the sampling distance, i.e. the distance between the sample surface and the cell inlet, in a dynamic manner. This distance was observed to influence the single pulse response profile. The variation of the average signal intensity at multiple sample heights within a range of 0.5 mm was <3% RSD. Under optimum conditions, single pulse responses with a full width at 10% of the maximum peak intensity (FW0.1M) of ∼1 ms can be achieved for 238U upon ablation of NIST SRM612 glass, effectively opening the way to pixel acquisition rates up to 1 kHz. To demonstrate the potential of this technology, the elemental distribution of Zn in small intestine villi of mice subjected to a Zn-enriched diet was imaged using the 0.6 µm spot size, and rapid imaging of a zircon grain cross-section was performed.

Laser Ablation-Inductively Coupled Plasma Time-of-Flight Mass Spectrometry Imaging of Trace Elements at the Single-Cell Level for Clinical Practice.

In this work, a combination of routine clinical practice and state-of-the-art laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) imaging is presented for multielement analysis of single cells on clinical samples. More specifically, routinely drawn blood thin films of a patient undergoing treatment with the anticancer drug cisplatin were studied. The presented label-free approach enabled rapid analysis of hundreds of cells at the single-cell level within a few minutes without additional tailored sample preparation. The employed low-dispersion LA setup is based on the tube-type COBALT ablation cell in combination with the aerosol rapid introduction system (ARIS) providing pixel-resolved imaging at 250-500 Hz for biological sample material. In order to cope with the short transient signals of only a few milliseconds delivered by the laser ablation setup, an icpTOF 2R TOF-based ICP-MS instrument was used for analysis, which has a mass coverage of m/ z = 14-256. Leukocytes and erythrocytes, imaged with a laser beam of 4 µm and pixel interspacing of 2 µm, were differentiated on the basis of their intrinsic trace-elemental pattern. Overall, red blood cells displayed high iron intensities, whereas individual white blood cells were characterized by their high phosphorus content and increased sulfur signal. Unsupervised multivariate statistical analysis was applied to the data set. Principal component plots showed a clear clustering of leukocytes versus erythrocytes. The approach allowed studying not only the drug distribution between plasma and cells but also, for the first time, the preferential accumulation of platinum in different blood cell types without the need of cell fixation and labeling. Extracellular hotspots of platinum were observed, whereas only a small fraction of platinum was associated with erythrocytes. The investigation demonstrates the potential of low-dispersion LA-ICP-TOFMS as a rapid and powerful tool for label-free single-cell imaging in the clinical context.

Three-dimensional reconstruction of the distribution of elemental tags in single cells using laser ablation ICP-mass spectrometry via registration approaches.

This paper describes a workflow towards the reconstruction of the three-dimensional elemental distribution profile within human cervical carcinoma cells (HeLa), at a spatial resolution down to 1 µm, employing state-of-the-art laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) instrumentation. The suspended cells underwent a series of fixation/embedding protocols and were stained with uranyl acetate and an Ir-based DNA intercalator. A priori, laboratory-based absorption micro-computed tomography (µ-CT) was applied to acquire a reference frame of the morphology of the cells and their spatial distribution before sectioning. After CT analysis, a trimmed 300 × 300 × 300 µm3 block was sectioned into a sequential series of 132 sections with a thickness of 2 µm, which were subjected to LA-ICP-MS imaging. A pixel acquisition rate of 250 pixels s-1 was achieved, through a bidirectional scanning strategy. After acquisition, the two-dimensional elemental images were reconstructed using the timestamps in the laser log file. The synchronization of the data required an improved optimization algorithm, which forces the pixels of scans in different ablation directions to be spatially coherent in the direction orthogonal to the scan direction. The volume was reconstructed using multiple registration approaches. Registration using the section outline itself as a fiducial marker resulted into a volume which was in good agreement with the morphology visualized in the µ-CT volume. The 3D µ-CT volume could be registered to the LA-ICP-MS volume, consisting of 2.9 × 107 voxels, and the nucleus dimensions in 3D space could be derived.

Correction to: Three-dimensional reconstruction of the distribution of elemental tags in single cells using laser ablation ICP-mass spectrometry via registration approaches.

The authors would like to call the reader's attention to the fact that unfortunately the originally provided affiliation for Dr. Tomoko Asaoka was not correct.

Imaging Artifacts in Continuous Scanning 2D LA-ICPMS Imaging Due to Nonsynchronization Issues.

Pulsed laser ablation (LA) devices in laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) imaging have become very advanced, delivering laser pulses with high temporal accuracy and stable energy density. However, unintentional imaging artifacts may be generated in 2D element maps when the LA repetition rate and the data acquisition parameters of ICPMS instruments with a sequential mass spectrometer (i.e., quadrupole filter or sector-field mass spectrometer) are desynchronized. This may potentially lead to interference patterns, visible as ripples in elemental images, and thus, compromised image quality. This paper describes the background of aliasing in continuous scanning mode through simulation experiments and ways to modulate the effect. The existence of this image degradation source is demonstrated experimentally via real-life imaging of a homogeneous glass standard.

Fast High-Resolution Laser Ablation-Inductively Coupled Plasma Mass Spectrometry Imaging of the Distribution of Platinum-Based Anticancer Compounds in Multicellular Tumor Spheroids.

Multicellular tumor spheroid models serve as an important three-dimensional in vitro cell model system as they mimic the complex tumor microenvironment and thus have contributed to valuable assays in drug discovery studies. In this study, we present a state-of-the-art laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) setup for high spatial resolution elemental imaging of multicellular tumor spheroids and an approach to account for variations in cell density. A low dispersion LA-ICPMS setup was employed, providing accelerated throughput and high sensitivity and permitting a lateral image resolution down to ∼2.5 µm for phosphorus and platinum in HCT116 colon cancer spheroids upon treatment with the clinically used anticancer drug oxaliplatin. Phosphorus was introduced as scalar to compensate for differences in cell density and tissue thickness and the Pt/P ratios together with the high resolution adopted in our approach allows the differentiation of platinum accumulation within each part of the morphology of the tumor spheroids (layers of proliferating, quiescent, and necrotic cells).

Three-Dimensional Reconstruction of the Tissue-Specific Multielemental Distribution within Ceriodaphnia dubia via Multimodal Registration Using Laser Ablation ICP-Mass Spectrometry and X-ray Spectroscopic Techniques.

In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 µm via a data fusion approach employing state-of-the-art laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) and laboratory-based absorption microcomputed tomography (µ-CT). C. dubia was exposed to elevated Cu, Ni, and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to µ-CT analysis. Subsequently, the sample was cut into 5 µm thin sections that were subjected to LA-ICP-TOFMS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOFMS images relative to the corresponding slices of the 3D µ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOFMS data were projected onto the µ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal µ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.

Quantitative Determination and Subcellular Imaging of Cu in Single Cells via Laser Ablation-ICP-Mass Spectrometry Using High-Density Microarray Gelatin Standards.

This manuscript describes the development and characterization of a high-density microarray calibration standard, manufactured in-house and designed to overcome the limitations in precision, accuracy, and throughput of current calibration approaches for the quantification of elemental concentrations on the cellular level using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). As a case study, the accumulation of Cu in the model organism Scrippsiella trochoidea resulting from transition metal exposure (ranging from 0.5 to 100 µg/L) was evaluated. After the Cu exposure, cells of this photosynthetic dinoflagellate were treated with a critical point drying protocol, transferred to a carbon stub, and sputter-coated with a Au layer for scanning electron microscopy (SEM) analysis. In subsequent LA-ICPMS analysis, approximately 100 cells of each population were individually ablated. This approach permitted the evaluation of the mean concentration of Cu in the cell population across different exposure levels and also allowed the examination of the cellular distribution of Cu within the populations. In a cross-validation exercise, subcellular LA-ICPMS imaging was demonstrated to corroborate synchrotron radiation confocal X-ray fluorescence (SR-XRF) microimaging of single cells investigated under in vivo conditions.

Submicrometer Imaging by Laser Ablation-Inductively Coupled Plasma Mass Spectrometry via Signal and Image Deconvolution Approaches.

In this work, pre- and postacquisition procedures for enhancing the lateral resolution of laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) in two- and three-dimensional (2D, 3D) nuclide distribution mapping beyond the laser beam waist are described. 2D images were constructed by projecting a rectangular grid of discrete LA positions, arranged at interspacings smaller than the dimensions of the laser beam waist, onto the sample surface, thus oversampling the region of interest and producing a 2D image convolved in the spatial domain. The pulse response peaks of a low-dispersion LA cell were isolated via signal deconvolution of the transient mass analyzer response. A 3D stack of 2D images was deconvolved by an iterative Richardson-Lucy algorithm with Total Variance regularization, enabling submicrometer image fidelity, demonstrated in the analysis of trace level features in corroded glass. A point spread function (PSF) could be derived from topography maps of single pulse craters from atomic force microscopy. This experimental PSF allows the approach to take into account the laser beam shape, beam aberrations, and the laser-solid interaction, which in turn enhances the spatial resolution of the reconstructed volume.

The Distribution and Biogenic Origins of Zinc in the Mineralised Tooth Tissues of Modern and Fossil Hominoids: Implications for Life History, Diet and Taphonomy.

Zinc is incorporated into enamel, dentine and cementum during tooth growth. This work aimed to distinguish between the processes underlying Zn incorporation and Zn distribution. These include different mineralisation processes, the physiological events around birth, Zn ingestion with diet, exposure to the oral environment during life and diagenetic changes to fossil teeth post-mortem. Synchrotron X-ray Fluorescence (SXRF) was used to map zinc distribution across longitudinal polished ground sections of both deciduous and permanent modern human, great ape and fossil hominoid teeth. Higher resolution fluorescence intensity maps were used to image Zn in surface enamel, secondary dentine and cementum, and at the neonatal line (NNL) and enamel-dentine-junction (EDJ) in deciduous teeth. Secondary dentine was consistently Zn-rich, but the highest concentrations of Zn (range 197-1743 ppm) were found in cuspal, mid-lateral and cervical surface enamel and were similar in unerupted teeth never exposed to the oral environment. Zinc was identified at the NNL and EDJ in both modern and fossil deciduous teeth. In fossil specimens, diagenetic changes were identified in various trace element distributions but only demineralisation appeared to markedly alter Zn distribution. Zinc appears to be tenacious and stable in fossil tooth tissues, especially in enamel, over millions of years.

Using SXRF and LA-ICP-TOFMS to Explore Evidence of Treatment and Physiological Responses to Leprosy in Medieval Denmark.

Leprosy can lead to blood depletion in Zn, Ca, Mg, and Fe and blood enrichment in Cu. In late medieval Europe, minerals were used to treat leprosy. Here, physiological responses to leprosy and possible evidence of treatment are investigated in enamel, dentine, and cementum of leprosy sufferers from medieval Denmark (n = 12) and early 20th century Romania (n = 2). Using SXRF and LA-ICP-TOFMS, 12 elements were mapped in 15 tooth thin sections, and the statistical covariation of paired elements was computed to assess their biological relevance. The results show marked covariations in the Zn, Ca, and Mg distributions, which are compatible with clinical studies but cannot be directly attributed to leprosy. Minerals used historically as a treatment for leprosy show no detectable intake (As, Hg) or a diffuse distribution (Pb) related to daily ingestion. Intense Pb enrichments indicate acute incorporations of Pb, potentially through the administration of Pb-enriched medication or the mobilization of Pb from bone stores to the bloodstream during intense physiological stress related to leprosy. However, comparisons with a healthy control group are needed to ascertain these interpretations. The positive correlations and the patterns observed between Pb and essential elements may indicate underlying pathophysiological conditions, demonstrating the potential of SXRF and LA-ICP-TOFMS for paleopathological investigations.

Impact of claudin-10 deficiency on amelogenesis: Lesson from a HELIX tooth.

In epithelia, claudin proteins are important components of the tight junctions as they determine the permeability and specificity to ions of the paracellular pathway. Mutations in CLDN10 cause the rare autosomal recessive HELIX syndrome (Hypohidrosis, Electrolyte imbalance, Lacrimal gland dysfunction, Ichthyosis, and Xerostomia), in which patients display severe enamel wear. Here, we assess whether this enamel wear is caused by an innate fragility directly related to claudin-10 deficiency in addition to xerostomia. A third molar collected from a female HELIX patient was analyzed by a combination of microanatomical and physicochemical approaches (i.e., electron microscopy, elemental mapping, Raman microspectroscopy, and synchrotron-based X-ray fluorescence). The enamel morphology, formation time, organization, and microstructure appeared to be within the natural variability. However, we identified accentuated strontium variations within the HELIX enamel, with alternating enrichments and depletions following the direction of the periodical striae of Retzius. These markings were also present in dentin. These data suggest that the enamel wear associated with HELIX may not be related to a disruption of enamel microstructure but rather to xerostomia. However, the occurrence of events of strontium variations within dental tissues might indicate repeated episodes of worsening of the renal dysfunction that may require further investigations.

Globally distributed iridium layer preserved within the Chicxulub impact structure.

The Cretaceous-Paleogene (K-Pg) mass extinction is marked globally by elevated concentrations of iridium, emplaced by a hypervelocity impact event 66 million years ago. Here, we report new data from four independent laboratories that reveal a positive iridium anomaly within the peak-ring sequence of the Chicxulub impact structure, in drill core recovered by IODP-ICDP Expedition 364. The highest concentration of ultrafine meteoritic matter occurs in the post-impact sediments that cover the crater peak ring, just below the lowermost Danian pelagic limestone. Within years to decades after the impact event, this part of the Chicxulub impact basin returned to a relatively low-energy depositional environment, recording in unprecedented detail the recovery of life during the succeeding millennia. The iridium layer provides a key temporal horizon precisely linking Chicxulub to K-Pg boundary sections worldwide.

Synchrotron X-ray fluorescence imaging of strontium incorporated into the enamel and dentine of wild-shot orangutan canine teeth.

OBJECTIVES: Dental hard tissues contain trace elements of both dietary and environmental origin. One objective was to demonstrate that a longitudinal record of synchronous Sr incorporation into enamel and dentine can be retrieved from museum specimens of once-free-living endangered species. Further objectives were to quantify sudden fluctuations in Sr concentration and estimate the extent of Sr overprinting back into dentine and enamel formed prior to the time of Sr ingestion. MATERIALS AND METHODS: Daily incremental markings were used to determine rates and times of tooth formation and synchrotron X-ray fluorescence of the same polished ground sections to image Sr distribution in a male and a female orangutan canine. The X-ray beam was monochromatised to 17.0 keV and focused to 500 × 500 nm2. Scans were performed at either 25.0 or 5.0 µm resolution. RESULTS: Baseline Sr levels ranged between 215-750 ppm. Multiple short, intense Sr labels reaching 750- 1,625 ppm occurred randomly throughout 15-22 years of tooth formation. In dentine, Sr concentration increased gradually away from the EDJ, while in enamel, it reduced towards the enamel surface. Using daily incremental markings, Sr overprinting into earlier formed dentine and enamel was estimated to be ∼12-45 days. There was no evidence of Sr overprinting by maturational ameloblasts. CONCLUSIONS: A good record of growth and trace element incorporation into tooth tissues can be retrieved from museum specimens. Short, intense Sr labels were equally well time-resolved in enamel and dentine and could be distinguished from more diffuse background levels. Enamel maturation appears to have no quantifiable effect.

High-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry for the determination of membranous receptor expression levels in breast cancer cell lines using receptor-specific hybrid tracers.

This work evaluates the possibility of placement of high-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) within precision medicine by assessing the suitability of LA-ICP-MS as a micro-analytical technique for the localization and quantification of membranous receptors in heterogeneous cell samples that express both the membrane-bound receptors C-X-C chemokine receptor type 4 (CXCR4) and epidermal growth factor receptor (EGFR). Staining of the breast cancer cell lines MDA-MB-231 X4 and MDA-MB-468 was achieved using receptor-specific hybrid tracers, containing both a fluorophore and a DTPA single-lanthanide chelate. Prior to LA-ICP-MS imaging, fluorescence confocal microscopy (FCM) imaging was performed to localize the receptors, hereby enabling direct comparison. Based on the different expression levels of CXCR4 and EGFR, a distinction could be made between the cell lines using both imaging modalities. Furthermore, FCM and LA-ICP-MS demonstrated complementary characteristics, as a more distinct discrimination could be made between both cell lines based on the EGFR-targeting hybrid tracer via LA-ICP-MS, due to the intrinsic CXCR4-related green fluorescent protein (GFP) signal present in the MDA-MB-231 X4 cells. Employing state-of-the-art LA-ICP-MS instrumentation in bidirectional area scanning mode for sub-cellular imaging of MDA-MB-231 X4 cells enabled the specific binding of the CXCR4-targeting hybrid tracer to the cell membrane to be clearly demonstrated. The stretching of cells over the glass substrate led to a considerably higher signal response for pixels at the cell edges, relative to the more central pixels. The determination of the expression levels of CXCR4 and EGFR for the MDA-MB-468 cell line was performed using LA-ICP-MS single-cell analysis (sc-LA-ICP-MS) and external calibration, based on the quantitative ablation of Ho-spiked dried gelatin droplet standards. Additionally, a second calibration approach was applied based on spot ablation of highly homogeneous dried gelatin gels in combination with the determination of the ablated volume using atomic force microscopy (AFM) and yielded results which were in good agreement with the expression levels determined via flow cytometry (FC) and mass cytometry (MC). Hybrid tracers enable a direct comparison between (i) FCM and LA-ICP-MS imaging for the evaluation of the microscopic binding pattern and between (ii) FC, MC and sc-LA-ICP-MS for the quantification of receptor expression levels in single cells.

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics.

BACKGROUND: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. METHODS: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). RESULTS: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. CONCLUSION: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods.

High-resolution laser ablation-inductively coupled plasma-mass spectrometry imaging of cisplatin-induced nephrotoxic side effects.

Two-dimensional elemental mapping (bioimaging) via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was performed on 5 µm thick formalin-fixed, paraffin-embedded kidney tissue sections from Cynomolgus monkeys administered with increasing pharmacological doses of cisplatin. Laterally resolved pixels of 1 µm were achieved, enabling elemental analysis on a (sub-)cellular level. Zones of high Pt response were observed in the renal cortex, where proximal tubules are present, the epithelium of which is responsible for partial reabsorption of cisplatin. Histopathological evaluation, of hematoxylin and eosin-stained serial sections, adjacent to the sections probed via LA-ICP-MS, revealed minimal to mild cisplatin-related lesions (<100 µm) in the renal cortex. Necrotic proximal tubules with sloughed epithelial cells in their lumen could be linked directly to the areas with the highest accumulation of cisplatin, indicating a direct link between cellular concentration and toxicity, thereby providing more insight into the mechanisms through which renal damage occurs.

Nanoscopic tumor tissue distribution of platinum after intraperitoneal administration in a xenograft model of ovarian cancer.

There is increasing interest in the treatment of advanced stage ovarian cancer (OC) using intraperitoneal (IP) delivery of platinum (Pt)-based chemotherapy. The antitumor efficacy of IP chemotherapy is determined by efficient tumor tissue penetration. Although it is assumed that Pt penetration is limited to a few millimeters after IP delivery, little is known on the distribution of Pt in different tumor compartments at the ultrastructural level following IP administration. Here, using synchrotron radiation X-ray fluorescence spectrometry (SR-XRF) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), Pt distribution and penetration in OC peritoneal xenografts were determined at nanometer scale after IP chemoperfusion of cisplatin at 37-38°C or 40-41°C (hyperthermic). Using principal component analysis (PCA) the presence of phosphorus, manganese, calcium, zinc, iron, bromine, and sulfur was correlated with the distribution of Pt, while k-means analysis was used to quantify the amount of Pt in weight% in tumor stroma and in tumor cells. The results showed a heterogeneous distribution of Pt throughout the tumor, with an accumulation in the extracellular matrix. LA-ICP-MS mappings indicated significantly higher concentrations of Pt (P=0.0062) after hyperthermic chemoperfusion of cisplatin, while SR-XRF demonstrated a deeper tissue Pt penetration after hyperthermic treatment. Using PCA, it was showed that Pt co-localizes with bromine and sulfur. No differences were observed in Pt distribution regarding tumor cells and stroma, when comparing normo- vs. hyperthermic treatment. In conclusion, SR-XRF and LA-ICP-MS are suitable and highly sensitive techniques to analyze the penetration depth and distribution of Pt-based drugs after IP administration. To the best of our knowledge, this is the first experiment in which the distribution of Pt is analyzed at the cellular level after IP administration of cisplatin.